ABSTRACT Toll-like receptors couple molecular recognition of conserved and structurally unique microbial molecules to rapid mobilization of innate immune effector systems and later induction of adaptive immunity. Toll-like receptor 4 (TLR4) is essential for the recognition of even minute amounts of endotoxin (E), unique glycolipids abundantly present on the surface of Gram negative bacteria (GNB), that are responsible for host responses to many GNB. Potent responses to E require the sequential action of several extracellular and cell surface host proteins, including lipopolysaccharide-binding protein (LBP), CD14, MD-2 for delivery to and activation of TLR4. Endotoxin-dependent responses elicited through TLR4 lead to activation of MyD88- and TRIF-dependent signaling pathway. Variations in endotoxin structure - as occurs naturally among GNB - and variations in levels of host cell proteins involved in E binding and signal transduction make possible great diversification of host cell responses to E. The normally protective responses to E may contribute to severe host pathology by exacerbating systemic inflammatory responses when local infection is not contained, as in GNB sepsis. Inappropriate signaling, whether too extensive or too modest, may contribute to a variety of infectious and inflammatory diseases including sepsis, atherosclerosis, autoimmune syndromes, liver disease, and acute lung injury. The novel reagents and experimental approaches we have developed allowed detection and quantitative analysis of specific, high affinity (pM) E- protein and TLR4 interactions under physiologically relevant conditions and so permitted determination of the mechanism of action of several natural and synthetic regulators of TLR4 activation. The discovery and purification of stable, water-soluble monomeric complexes of E.MD-2 that, depending on the structure of bound E or MD-2, can act at pM concentrations as TLR4 agonists or antagonists, have provided unique reagents to probe the remaining mysteries of endotoxin-triggered TLR4 activation and the structural limits of pattern recognition of endotoxin by MD-2 and TLR4. Increasing evidence suggests physiologic and patho-physiologic roles of TLR4 extending beyond host responses to invading GNB. Thus, knowledge of the molecular and cellular rules regulating TLR4 function should provide new insights into what determines the nature and the strength of TLR4-dependent responses in settings including but not limited to GNB infection. The long-term goals of our work are to better understand the mechanism(s) by which TLR4-dependent cellular responses to GNB endotoxin and host-derived danger molecules are regulated. More specifically, the work is focused on understanding 1) the mechanism(s) by which E interactions with MD-2 specify activation or antagonism of TLR4 and the contrasting structural and molecular requirements for hemin activation of TLR4; 2) the effects of the naturally occurring polymorphic variant substitutions D299G and/or T399I in the TLR4 ectodomain on surface expression and function of TLR4; and 3) the effects of variables in E (lipid A) structure and presentation on induction of MyD88- vs. TRIF-dependent signaling by activated TLR4. The two pathways are linked to different cellular responses so a better understanding of their differential regulation could have important implications for the design of compounds such as adjuvants with selective effects on the type and duration of TLR4 signaling. The studies proposed here should significantly increase understanding of the regulation of TLR4 function and produce insights applicable to the design and testing of novel TLR4-directed immune-modulators. Knowledge of the molecular and cellular rules regulating TLR4 function contributes to the understanding of what determines the nature and the strength of TLR4-dependent responses in settings including, but not limited to, GNB infection.